This project focuses on the biogenesis of Lamin A, a critical structural component of the nuclear envelope. Surprisingly, recent studies have revealed that mutations in the gene encoding Lamin A result in a wide range of diseases called the "laminopathies" (encompassing cardiomyopathy, muscular dystrophy, lipodystrophy, and progeroid disorders), leading to a resurgence of interest in the biology of Lamin A. The Lamin A precursor, prelamin A, undergoes a series of post-translational processing events, including: 1) C- terminal CaaX modification (prenylation, proteolysis, and carboxyl methylation), followed by 2) an endoproteolytic cleavage event, mediated by the zinc metalloprotease ZmpSte24, that removes the CaaX- modified C-terminus to yield mature Lamin A. In previous years of this project, our laboratory discovered ZmpSte24 as a key enzyme in the biogenesis of yeast a-factor. From these findings this project has evolved to study the biogenesis of Lamin A in mammalian cells. The most severe laminopathy is the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS). Strikingly, there appears to be a direct link between defective Zmpste24-mediated endoproteolytic processing of prelamin A and progeroid diseases based on the findings that: 1) HGPS results from a mutation in which the ZmpSte24 cleavage site within Lamin A is deleted, and 2) the progeroid disorders mandibuloacral dysplasia (MAD) and restrictive dermopathy (RD) map to ZmpSte24. Thus, it appears that the persistently prenylated form of Lamin A that is present in HGPS or zmpste24-/- cells leads to accelerated aging pathologies. In this proposal we will define key cell biological aspects of Lamin A processing and address the role that a lack of processing plays in disease mechanisms. We will use molecular, cell biological, genetic, and biochemical approaches to address the following aims: Aim 1) determine the cellular location of Lamin A processing (nucleoplasmic vs. the cytosolic face of the ER);Aim 2) determine the fate of the cleaved C- terminal tail;Aim 3) determine the recognition sequences within prelamin A important for ZmpSte24 cleavage, and define how Zmpste24 cleaves prelamin A using purified enzyme;and Aim 4) investigate the molecular mechanisms by which failure to cleave the prelamin A tail leads to cellular and disease phenotypes. In particular we will test whether methylation may contribute to the toxicity of Lamin A in HGPS. The intriguing finding that progeroid diseases are caused by incomplete processing of prelamin A has underscored the importance of a comprehensive understanding of the entire processing pathway, which we address in this proposal. Our studies will provide insight into therapeutic options for progeroid disorders. The significance of this research is heightened by recent findings that inhibition of ZmpSte24-mediated processing of prelamin A may contribute to HIV therapy-induced side effects, and possibly to the mechanisms of normal aging. PUBLIC HEALTH RELEVANCE: Mutations in the nuclear structural protein Lamin A cause the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS) and a spectrum of diseases known as "laminopathies". This project addresses fundamental unanswered questions about Lamin A biology, including how it is processed within the cell and how abnormal processing can cause disease, as in HGPS. Our studies will provide insight into potential therapeutic options for premature aging disorders, and may also shed light on the mechanisms underlying the normal aging process.